CN1214824C - Method of regulating degradation rate of porous collagen-based cradle with amino acid - Google Patents
Method of regulating degradation rate of porous collagen-based cradle with amino acid Download PDFInfo
- Publication number
- CN1214824C CN1214824C CN 03117083 CN03117083A CN1214824C CN 1214824 C CN1214824 C CN 1214824C CN 03117083 CN03117083 CN 03117083 CN 03117083 A CN03117083 A CN 03117083A CN 1214824 C CN1214824 C CN 1214824C
- Authority
- CN
- China
- Prior art keywords
- collagen
- quito
- hole
- amino acid
- support
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 71
- 108010035532 Collagen Proteins 0.000 title claims abstract description 71
- 229920001436 collagen Polymers 0.000 title claims abstract description 71
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 34
- 230000015556 catabolic process Effects 0.000 title claims abstract description 34
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 230000001276 controlling effect Effects 0.000 claims abstract description 8
- 235000001014 amino acid Nutrition 0.000 claims description 31
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 16
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 16
- 239000004472 Lysine Substances 0.000 claims description 15
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 9
- -1 carbodiimides compounds Chemical class 0.000 claims description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 8
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 5
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 claims description 4
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 claims description 4
- 108090000723 Insulin-Like Growth Factor I Chemical class 0.000 claims description 4
- 102000013275 Somatomedins Human genes 0.000 claims description 4
- 229920001282 polysaccharide Polymers 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 239000004475 Arginine Substances 0.000 claims description 2
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 claims description 2
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 claims description 2
- 229920001661 Chitosan Polymers 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 claims description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 claims description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 229940112869 bone morphogenetic protein Drugs 0.000 claims description 2
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 2
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 abstract description 11
- 210000002435 tendon Anatomy 0.000 abstract description 8
- 210000001519 tissue Anatomy 0.000 abstract description 8
- 239000003795 chemical substances by application Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 5
- 230000008929 regeneration Effects 0.000 abstract description 4
- 238000011069 regeneration method Methods 0.000 abstract description 4
- 210000000056 organ Anatomy 0.000 abstract description 3
- 210000003491 skin Anatomy 0.000 abstract description 3
- 210000004204 blood vessel Anatomy 0.000 abstract description 2
- 210000000988 bone and bone Anatomy 0.000 abstract description 2
- 150000001718 carbodiimides Chemical class 0.000 abstract description 2
- 210000000845 cartilage Anatomy 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 210000003709 heart valve Anatomy 0.000 abstract description 2
- 210000005036 nerve Anatomy 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 230000017423 tissue regeneration Effects 0.000 abstract description 2
- 230000033667 organ regeneration Effects 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 235000018977 lysine Nutrition 0.000 description 19
- 102000012422 Collagen Type I Human genes 0.000 description 13
- 108010022452 Collagen Type I Proteins 0.000 description 13
- 210000002950 fibroblast Anatomy 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 230000002255 enzymatic effect Effects 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 7
- 235000015278 beef Nutrition 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000003431 cross linking reagent Substances 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 5
- 150000002669 lysines Chemical class 0.000 description 5
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 4
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229960002591 hydroxyproline Drugs 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- 230000006399 behavior Effects 0.000 description 3
- 230000008602 contraction Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- ROSDSFDQCJNGOL-UHFFFAOYSA-N protonated dimethyl amine Natural products CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 3
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Images
Landscapes
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
The present invention discloses a method for regulating and controlling the degradation rate of a collagen base porous stent by amino acid. Amino acid with good biocompatibility is used as a crosslinking and bridging agent by the method, amino acid of 0.1 to 100 muM is compounded with the collagen base porous stent so that a carbodiimide class compound is adopted for crosslinking treatment, and the collagen base porous stent with a controllable degradation rate and good biocompatibility is prepared. The method of the present invention has the advantages of no toxic or side effect of used materials, wide sources, low cost, simple and feasible preparation technology, mild conditions and good repeatability. The obtained porous stent can satisfy different requirements of tissue and organ regeneration, can be widely applied to the regeneration and the reconstruction of organs, such as skin, cartilage, bones, blood vessels, nerves, tendons, heart valves, etc., and has wide application value in the fields of biomedicine, tissue engineering, etc.
Description
Technical field
The present invention relates to aminoacid is crosslinked bridging agent, the method for regulation and control hole, collagen Quito scaffold degradation speed under the effect of cross-linking agent.
Background technology
It is multidisciplinary, multi-field that organizational project relates to medical science, chemistry, biology, materialogy etc.In the research of organizational project, the selection of timbering material and the structure of support are key links wherein.Collagen is the main component in the mammal connective tissue, constitutes the protein of human body about 30%, and the dry weight in skin reaches 72%.Collagen has 19 types, and modal is I type, II type and III type.Wherein the I type is the abundantest, and function admirable, is widely used in biomaterial.Though collagen-based materials has unrivaled biocompatibility, the mechanical strength of the support that makes up with pure collagen is lower, and degradation rate is too fast, can not satisfy the requirement of tissue engineering bracket.Therefore be necessary by suitable method crosslinked improving the degree of cross linking of hole, collagen Quito support, and the degradation rate that is complementary of acquisition and tissue regeneration.At present, the cross-linking method of hole, collagen Quito support mainly contains physical crosslinking and chemical crosslinking.The physical crosslinking method mainly contains xeothermic crosslinked, ultraviolet-crosslinkable of vacuum etc.The characteristics of these class methods are not introduce other material in cross-linking process, can not destroy hole, collagen Quito support excellent biological compatibility, but simple physical crosslinking can not reach the desired degree of cross linking, and the degradation rate of collagen scaffold is still too fast, can not be complementary with the regeneration rate of tissue.Chemical crosslink technique is used more widely, and chemical cross-linking agent mainly contains glutaraldehyde, Carbodiimides and bis-epoxy compounds.The shortcoming of traditional pure chemistry cross-linking method is relatively more difficult usually for the regulation and control of the degree of cross linking, can not satisfy the requirement that different various tissues of regeneration rate and organ make up; During with glutaraldehyde as cross linker, the residual meeting of glutaraldehyde causes some side effect, as cytotoxicity.Aminoacid is proteinic basic structural unit, has excellent biological compatibility.And have two carboxyls or two amino in acidic amino acid or the basic amino acid molecule respectively, therefore in collagen base support crosslinked, can be used as crosslinked bridging agent.
Summary of the invention
The purpose of this invention is to provide a kind of method of regulating and control hole, collagen Quito scaffold degradation speed with aminoacid as crosslinked bridging agent.
Method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed of the present invention, its step is earlier hole, collagen Quito to be propped up to be placed in the Freamine that concentration is 0.1～100 μ M to soak 0.01～100 hour, then with compound hole, amino acid whose collagen Quito support in 1～1000mM carbodiimides compounds, carrying out crosslinked 1～48 hour under 0～100 ℃, get final product.
By changing amino acid whose kind, the concentration of amino acid whose ratio and cross-linking agent can be regulated and control the degradation rate of hole, collagen Quito support.
Among the present invention, the aminoacid kind is not had special requirement, adopt lysine, arginine, glutamic acid, aspartic acid etc. usually.The carbodiimides compounds is not had special requirement yet, but preferably adopt water-soluble carbodiimide, as 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides (EDAC).Hole, collagen Quito support is meant pure collagen or the compound porous support of other composition, as compound aminoacid, and polysaccharide compound or the compound porous support of somatomedin compounds, said polysaccharide compound is as chitosan, chondroitin sulfate.The somatomedin compounds is as bone morphogenetic protein, fibroblast growth factor, endothelial cell growth factor (ECGF), transforming growth factor etc.Said collagen source is not had special requirement, can be beef tendon collagen, pigskin collagen or Mus tail collagen etc.
The used crosslinked bridging agent of the inventive method has excellent biological compatibility and degradation property, has no side effect, and wide material sources, and with low cost, preparation is simple, mild condition, good reproducibility.Invention is crosslinked bridging agent with aminoacid, by changing amino acid whose kind, the concentration of amino acid whose ratio and cross-linking agent realizes the degradation rate of regulation and control hole, collagen Quito support, the porous support of gained can satisfy the needs of different tissues and neomorph, can be widely used in the regeneration and the reconstruction of organs such as skin, cartilage, bone, blood vessel, nerve, tendon and cardiac valve, be with a wide range of applications in fields such as biomedical and organizational projects.
Description of drawings
Fig. 1 is for adding the influence of variety classes aminoacid to hole, collagen Quito scaffold degradation degree.Support the type i collagen enzymatic solution of 0.1mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 2 is the variation relation of the degree of degradation of hole, collagen Quito support with aminoglutaric acid concentration.Support the type i collagen enzymatic solution of 0.5mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 3 is the variation relation of the degree of degradation of hole, collagen Quito support with lysine concentration.Support the type i collagen enzymatic solution of 0.5mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 4 is the variation relation of the palliating degradation degree of hole, collagen Quito support with 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides N-maloyl imines (EDAC/NHS) concentration.Support the type i collagen enzymatic solution of 0.1mg/ml (PBS, pH7.4) in, the results behind 37 ℃ of digestion 12h.
Fig. 5 is for the contrast collagen scaffold and add the collagen Quito hole support of 2.5 μ M lysines after crosslinked, the external contraction behavior under the fibroblast effect.Cell inoculation density is 500 * 10
4/ ml.
Fig. 6 is for adding the crosslinked laser confocal microscope photo of collagen scaffold (rhodamine labelling) plantation fibroblast after 3 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml, fluorescein(e) diacetate (FDA) dyeing.
Fig. 7 a is for adding crosslinked scanning electron microscope (SEM) photo of collagen scaffold plantation fibroblast after 7 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml.
Fig. 7 b is for adding the crosslinked SEM photo of collagen scaffold plantation fibroblast after 21 days of 2.5 μ M lysines.Cell inoculation density is 500 * 10
4/ ml.
Fig. 8 is for adding the crosslinked tissue slice photo (H﹠amp of collagen scaffold plantation fibroblast after 21 days of 2.5 μ M lysines; E dyeing).Cell inoculation density is 500 * 10
4/ ml.
The specific embodiment
The present invention may be better understood by following embodiment, but these examples are not used for limiting the present invention.
Different types of aminoacid is to the influence of the degree of degradation of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add 2-N-morpholino ethane sulfonic acid (MES) solution (50mM of glycine (Gly), glutamic acid (Glu) or lysine (Lys) that 1ml contains 2.5 μ M respectively, pH is 5.5), soaked 1 hour; Soak in contrast to add tri-distilled water simultaneously.Add 1ml then respectively and contain 1-ethyl-3-(dimethyl amine propyl group)-carbodiimides (EDAC) of 40mM, the MES solution of the N-maloyl imines (NHS) of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain hole, the collagen Quito support after crosslinked.
Get uncrosslinked, do not add aminoacid crosslinked, add crosslinked hole, the collagen Quito support of different aminoacids, (PBS pH7.4), digests 12h in 37 ℃ of thermostatic water bath to add the type i collagen enzymatic solution of 0.1mg/ml of 3ml.Draw digestion supernatant 1ml, place polymerization pipe, add the hydrochloric acid of the 6M of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.Degree of degradation uncrosslinked, that do not add the porous support that aminoacid is crosslinked, the interpolation different aminoacids is crosslinked is seen Fig. 1.
The concentration of glutamic acid is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of 1～10 μ M glutamic acid of 1ml respectively, soaked 1 hour; Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain hole, the collagen Quito support after crosslinked.
Get and add the crosslinked porous support of variable concentrations glutamic acid, (PBS pH7.4), digests 12h to the type i collagen enzymatic solution of the 0.5mg/ml of adding 3ml in 37 ℃ of thermostatic water bath.Draw digestion supernatant 1ml, place polymerization pipe, add the 6M hydrochloric acid of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of glutamic acid is seen Fig. 2 to the influence of the degree of degradation of hole, collagen Quito support.
Embodiment 3
The concentration of lysine is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of 0.67～100 μ M lysine of 1ml respectively, soaked 1 hour; Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get and add the crosslinked porous support of variable concentrations lysine, (PBS pH7.4), digests 12h to the type i collagen enzymatic solution of the 0.5mg/ml of adding 3ml in 37 ℃ of thermostatic water bath.Draw digestion supernatant 1ml, place polymerization pipe, add the 6M hydrochloric acid of 2ml, behind the tube sealing in 120 ℃ of hydrolysis 12 hours.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of lysine is seen Fig. 3 to the influence of the degree of degradation of hole, collagen Quito support.
Embodiment 4
The cross-linking agent of variable concentrations is to the influence of the degradation rate of hole, collagen Quito support.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of the lysine of 2.5 μ M respectively, soaked 1 hour; (50/25～5/2.5mM) EDAC/NHS solution, under room temperature crosslinked 24 hours, crosslinked back was with tri-distilled water rinsing 6 times, each 10 minutes to add the variable concentrations of 1ml then respectively.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get the three-dimensional rack 2.5mg after crosslinked, the type i collagen enzymatic solution (being made into PBS) that adds the 0.1mg/ml of 3ml then digests in 37 ℃ of thermostatic water bath, to required time absorption digestion supernatant 1ml, place polymerization pipe, the 6M hydrochloric acid that adds 2ml, hydrolysis 12 hours in 120 ℃ baking oven behind the tube sealing.The content of the hydroxyproline by measuring hydrolyzed solution characterizes the degree of degradation of collagen porous support.The concentration of cross-linking agent is seen Fig. 4 to the influence of the degree of degradation of hole, collagen Quito support.
The interpolation of lysine is to the influence of the external contraction behavior of hole, collagen Quito support under the fibroblast effect.
The type i collagen support (2.5mg/ piece) that derives from beef tendon is placed 24 well culture plates, add the MES solution (50mM, pH are 5.5) of lysine of the 2.5 μ M of 1ml respectively, soaked 1 hour, do not add amino acid whose sample with corresponding M ES solution soaking 1 hour.Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get uncrosslinked, do not add aminoacid crosslinked, add the crosslinked three-dimensional rack of lysine, plant human fibroblasts (5,000,000/ml), 37 ℃ of CO of 5% down of 400 μ l respectively
2Cultivate in the incubator, the next day change liquid.After having cultivated 3,7,14 and 21 days, measure the diameter of various supports respectively.Fig. 5 is the external contraction behaviors of various supports under the fibroblast effect.
Add the cell in vitro evaluation of hole, the collagen Quito support of lysine
The collagen scaffold (2.5mg/ piece) of getting the rhodamine labelling places 24 well culture plates, adds the MES solution (50mM, pH are 5.5) of the lysine of 1ml concentration 2.5 μ M respectively, soaks 1 hour.Add the EDAC that 1ml contains 40mM then respectively, the MES solution of the NHS of 40mM makes the concentration of final EDAC and NHS be respectively 20mM and 10mM.At room temperature crosslinked 24 hours, tri-distilled water rinsing 6 times of crosslinked back, each 10 minutes.Pass through freezing-lyophilization again, promptly obtain crosslinked hole, collagen Quito support.
Get and add the crosslinked collagen porous support of lysine, plant human fibroblasts (5% the CO under 5,000,000/ml), 37 ℃ of 400 μ l
2Cultivate in the incubator, the next day change liquid.After having cultivated 3 days, use fluorescein diacetate (FDA) dyeing back laser confocal microscope (CLSM) and observe respectively.As seen from Figure 6, fibroblast (left side) the plantation 3 days after can along collagen scaffold (in) hole wall sprawl, present good growth conditions (right side).Fig. 7 a and Fig. 7 b are respectively scanning electron microscope (SEM) photo of cultivating after 7 days and 21 days.Visible fibroblast is attached on the collagen and sprawls among the figure, presents typical fusiformis.Fig. 8 as seen from the figure at internal stent, has a large amount of fibroblasts to grow into, and is evenly distributed in support everywhere for the tissue slice figure of the support of cultivation after 21 days.
Claims (5)
1. method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed, it is characterized in that earlier hole, collagen Quito being propped up to be placed in the Freamine that concentration is 0.1～100 μ M soaking 0.01～100 hour, then compound hole, amino acid whose collagen Quito support was being carried out in 1～1000mM carbodiimides compounds crosslinked 1～48 hour under 0～100 ℃.
2. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that said aminoacid is lysine, arginine, glutamic acid, glycine or aspartic acid.
3. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that hole, said collagen Quito support is meant pure collagen porous support or compound hole, the collagen Quito support of aminoacid, polysaccharide compound or somatomedin compounds.
4. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 3, it is characterized in that polysaccharide compound is chitosan or chondroitin sulfate, the somatomedin compounds is bone morphogenetic protein, fibroblast growth factor, endothelial cell growth factor (ECGF) or transforming growth factor.
5. the method with hole, amino acid regulating and controlling collagen Quito scaffold degradation speed according to claim 1 is characterized in that said carbodiimides compounds is a water-soluble carbodiimide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03117083 CN1214824C (en) | 2003-05-19 | 2003-05-19 | Method of regulating degradation rate of porous collagen-based cradle with amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03117083 CN1214824C (en) | 2003-05-19 | 2003-05-19 | Method of regulating degradation rate of porous collagen-based cradle with amino acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1453044A CN1453044A (en) | 2003-11-05 |
| CN1214824C true CN1214824C (en) | 2005-08-17 |
Family
ID=29260086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03117083 Expired - Fee Related CN1214824C (en) | 2003-05-19 | 2003-05-19 | Method of regulating degradation rate of porous collagen-based cradle with amino acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1214824C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107998453B (en) * | 2017-12-12 | 2020-09-25 | 中山大学附属第一医院 | Surface-modified acellular matrix and modification method thereof |
-
2003
- 2003-05-19 CN CN 03117083 patent/CN1214824C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1453044A (en) | 2003-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Adamiak et al. | Current methods of collagen cross-linking | |
| Riaz et al. | FTIR analysis of natural and synthetic collagen | |
| Kato et al. | Mechanical properties of collagen fibres: a comparison of reconstituted and rat tail tendon fibres | |
| Verma et al. | Preparation of scaffolds from human hair proteins for tissue-engineering applications | |
| DE68916900T2 (en) | EXCITATION OF CHEMOTAXE BY CHEMOTACTIC PEPTIDES. | |
| US4980403A (en) | Collagen products | |
| US20160194378A1 (en) | Peptides and uses thereof | |
| Veeruraj et al. | Isolation and characterization of drug delivering potential of type-I collagen from eel fish Evenchelys macrura | |
| US20040136977A1 (en) | Crosslinked elastin and process for producing the same | |
| Yamauchi et al. | Enhanced cell adhesion on RGDS-carrying keratin film | |
| Nwe et al. | Selection of a biopolymer based on attachment, morphology and proliferation of fibroblast NIH/3T3 cells for the development of a biodegradable tissue regeneration template: Alginate, bacterial cellulose and gelatin | |
| CN107630060B (en) | Self-assembled collagen and preparation method thereof | |
| CN1233425C (en) | Method for layer-layer self-assembling and fixing biological active factor on tissue engineering material | |
| US9018009B2 (en) | Thermally induced gelation of collagen hydrogel and method of thermally inducing gelling a collagen hydrogel | |
| CN1179758C (en) | Preparation method of collagen/chitosan porous scaffold for tissue engineering | |
| Huang et al. | Origin of critical nature and stability enhancement in collagen matrix based biomaterials: Comprehensive modification technologies | |
| CN109762184B (en) | Preparation method of collagen hydrogel | |
| JP5875761B2 (en) | Collagen fiber gel and use thereof | |
| CN107630059B (en) | Novel self-assembled collagen and preparation method thereof | |
| CN115887742A (en) | Preparation method of antibacterial functional collagen-based injectable self-repairing hydrogel | |
| CN107412861A (en) | The Bone Defect Repari gel of recombined collagen sulfate composite chondroitin and polyethylene glycol | |
| CN1214824C (en) | Method of regulating degradation rate of porous collagen-based cradle with amino acid | |
| CN1486753A (en) | Prepn of injectable bone-repairing hydrochlorinated chitosan/calcium phosphate material | |
| JP2021078642A (en) | Vitrigel | |
| JPH11228837A (en) | Silk protein/collagen composite material and its production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |